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Applied and Environmental Microbiology, December 2001, p. 5593-5600, Vol. 67, No. 12
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.12.5593-5600.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Detection of Hepatitis A Virus by the Nucleic Acid Sequence-Based Amplification Technique and Comparison with Reverse Transcription-PCR

Julie Jean,1 Burton Blais,2 André Darveau,3 and Ismaïl Fliss1,*

Centre de Recherche STELA, Département de Sciences des Aliments et de Nutrition,1 Département de Biochimie et Microbiologie,3 Université Laval, Québec G1K 7P4, and Canadian Food Inspection Agency, Laboratory Services Division, Ottawa K1A 0C6,2 Canada

Received 18 May 2001/Accepted 26 September 2001

A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 × 102 PFU/ml) was obtained with NASBA, compared to 50 PFU (1 × 104 PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

* Corresponding author. Mailing address: Centre de Recherche STELA, Département de Sciences des Aliments et de Nutrition, Université Laval, Québec (QC) G1K 7P4, Canada. Phone: 418-656-2131, ext. 6825. Fax: 418-656-3353. E-mail: ismail.fliss{at}aln.ulaval.ca.

Applied and Environmental Microbiology, December 2001, p. 5593-5600, Vol. 67, No. 12
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.12.5593-5600.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.